Electron microscopic studies of the endocytotic process of cationized ferritin in cultured human retinal pigment epithelial cells

Summary The endocytotic process in cultured human RPE cells was observed after 1 min, 20 min, and 2 h incubation with cationized ferritin. Within 1 min the ferritin particles were seen to attach to the cell membrane, especially between microvilli. Uncoated and coated pits could be recognized on the cell membranes, and uncoated and coated endocytotic vesicles were found in the cytoplasm after 20 min of incubation. These vesicles were surrounded by abundant microfilaments and had no visible membranes. Loss of membrane may be an initial step in the process of developing into the irregular clumps of ferritin particles found inside the plasma membrane. With time, more irregular clumps of ferritin, smaller than the particles introduced during incubation, appeared just beneath the cell membrane. Lysosomes were adjacent to the clumps of ferritin particles associated with microtobules and finally degraded these particles. The phagolysosomes containing many particles were surrounded by many microtubules. Small ferritin particles surrounded but had not entered the rough endoplasmic reticulums, and no particles were seen either around or in the Golgi apparatus. Presented at the 7th International Congress of Eye Research, Nagoya, Japan, 27 September 1986.     

Some factors controlling cell polarity in chick retinal pigment epithelial cells in clonal culture

In clonal culture differentiated chick retinal pigmented epithelial (RPE) cells form a monolayer which shows little or no cellular division. The cells usually rest on a basal and reticular lamina and are polarized with their apical surface towards the medium. The apical surface is characterized by apical protrusions, an extensive apical web of microfilaments and junctional complexes which join the apical-lateral borders. A PA/S positive material with a felt-like appearance from the serum component of the medium coats the surfaces of the tissue culture plates. A similar material is found on any membrane filter which has been exposed to medium containing serum. When such a filter brought in contact with the upper surfaces of the RPE cells, the apical surface characteristics are lost, the cells often accumulate Alcian Blue positive material between the cells and the filter and secrete a reticular and a basal lamina, i.e. they establish a second basal surface. Once this has occurred, the cells appear to either detach from the plate and reverse their polarity, or undergo division forming two cell layers. In the latter case new apical surfaces are created between the cell layers but the cells appear to join to form circular structures rather than sheets. These results suggest that contact with this felt-like material initiates formation of a basal surface. They further suggest that where the apical surface has been converted to a basal one the cell attempts to restore the apical surface either by separating from the plate and reversing its polarity or by creating circular structures and developing new apices oriented toward the center of the circle.     

Role of integrins in differentiation of chick retinal pigmented epithelial cells in vitro

When retinal pigmented epithelial cells (PEC) of chick embryos are cultured under appropriate conditions, the phenotype changes to that of lens cells through a process known as transdifferentiation. The first half of the process, characterized by dedifferentiation of PEC, is accompanied by a marked decrease in adhesiveness of PEC to collagen type I- or type IV-coated dishes. To understand the underlying mechanisms of this change, we analyzed the expression of integrins, which are major receptors for extracellular matrix components. Northern blot analysis with cDNA probes for chicken α3, α6, α8, αv, β1 and β5 integrin mRNA showed that the genes for all these integrins are transcribed at similar levels in PEC and dedifferentiated PEC (dePEC). Further analysis of β1 integrin, which is a major component of integrin heterodimers, showed that although the protein amount of β1 integrin was not changed, its localization at focal contacts seen in PEC was lost in dePEC. When anti-β1 integrin antibody was added to the PEC culture medium, a decrease of cell-substrate adhesiveness occurred, followed by a gradual change in both morphology and gene expression patterns to ones similar to those of dePEC. These findings suggest that an appropriate distribution of β1 integrin plays an essential role in maintaining the differentiated state of PEC through cell-substrate adhesion.     

[32P]orthophosphate and [35S]methionine label separate pools of neurofilaments with markedly different axonal transport kinetics in mouse retinal ganglion cells in vivo

Newly synthesized neurofilament proteins become highly phosphorylated within axons. Within 2 days after intravitreously injecting normal adult mice with [³²P]orthophosphate, we observed that neurofilaments along the entire length of optic axons were radiolabeled by a soluble³²P-carrier that was axonally transported faster than neurofilaments.³²P-incorporation into neurofilament proteins synthesized at the time of injection was comparatively low and minimally influenced the labeling pattern along axons.³²P-incorporation into axonal neurofilaments was considerably higher in the middle region of the optic axons. This characteristic non-uniform distribution of radiolabel remained nearly unchanged for at least 22 days. During this interval, less than 10% of the total³²P-labeled neurofilaments redistributed from the optic nerve to the optic tract. By contrast, newly synthesized neurofilaments were selectively pulse-labeled in ganglion cell bodies by intravitreous injection of [³⁵S]methionine and about 60% of this pool translocated by slow axoplasmic transport to the optic tract during the same time interval. These findings indicate that the steady-state or resident pool of neurofilaments in axons is not identical to the newly synthesized neurofilament pool, the major portion of which moves at the slowest rate of axoplasmic transport. Taken together with earlier studies, these results support the idea that, depending in part on their phosphorylation state, transported neurofilaments can interact for short or very long periods with a stationary but dynamic neurofilament lattice in axons.Special issue dedicated to Dr. Sidney Ochs.     

Thioredoxin 1 overexpression attenuated diabetes-induced endoplasmic reticulum stress in Müller cells via apoptosis signal-regulating kinase 1

As one of the common and serious chronic complications of diabetes mellitus (DM), the related mechanism of diabetic retinopathy (DR) has not been fully understood. Müller cell reactive gliosis is one of the early pathophysiological features of DR. Therefore, exploring the manner to reduce diabetes-induced Müller cell damage is essential to delay DR. Thioredoxin 1 (Trx1), one of the ubiquitous redox enzymes, plays a vital role in redox homeostasis via protein–protein interactions, including apoptosis signal-regulating kinase 1 (ASK1). Previous studies have shown that upregulation of Trx by some drugs can attenuate endoplasmic reticulum stress (ERS) in DR, but the related mechanism was unclear. In this study, we used DM mouse and high glucose (HG)-cultured human Müller cells as models to clarify the effect of Trx1 on ERS and the underlying mechanism. The data showed that the diabetes-induced Müller cell damage was increased significantly. Moreover, the expression of ERS and reactive gliosis was also upregulated in diabetes in vivo and in vitro. However, it was reversed after Trx1 overexpression. Besides, ERS-related protein expression, reactive gliosis, and apoptosis were decreased after transfection with ASK1 small-interfering RNA in stable Trx1 overexpression Müller cells after HG treatment. Taken together, Trx1 could protect Müller cells from diabetes-induced damage, and the underlying mechanism was related to inhibited ERS via ASK1.     

Changes associated with tyrosine phosphorylation during short-term hypoxia in retinal microvascular endothelial cells in vitro

The occlusion of capillary vessels results in low oxygen tension in adjacent tissues which triggers a signaling cascade that culminates in neovascularization. Using bovine retinal capillary endothelial cells (BRCEC), we investigated the effects of short-term hypoxia on DNA synthesis, phosphotyrosine induction, changes in the expression of basic fibroblast growth factor receptor (bFGFR), protein kinase C (PKCα), heat shock protein 70 (HSP70), and SH2-containing protein (SHC). The effect of protein tyrosine kinase (PTK) and phosphatase inhibitors on hypoxia-induced phosphotyrosine was also studied. Capillary endothelial cells cultured in standard normoxic (pO₂ = 20%) conditions were quiesced in low serum containing medium and then exposed to low oxygen tension or hypoxia (pO₂ = 3%) in humidified, 5% CO₂, 37°C, tissue culture chambers, on a time-course of up to 24 h. DNA synthesis was potentiated by hypoxia in a time-dependent manner. This response positively correlated with the cumulative induction of phosphotyrosine and the downregulation of bFGFR (M_r ～ 85 kDa). Protein tyrosine kinase inhibitors, herbimycin-A, and methyl 2,5-dihydroxycinnamate, unlike genistein, markedly blocked hypoxia-induced phosphotyrosine. Prolonged exposure of cells to phosphatase inhibitor, sodium orthovanadate, also blocked hypoxia-induced phosphotyrosine. The expression of HSP70, PKCα, and SHC were not markedly altered by hypoxia. Taken together, these data suggest that short-term hypoxia activates endothelial cell proliferation in part via tyrosine phosphorylation of cellular proteins and changes in the expression of the FGF receptor. Thus, endothelial cell mitogenesis and neovascularization associated with low oxygen tension may be controlled by abrogating signaling pathways mediated by protein tyrosine kinase and phosphatases. © 1995 Wiley-Liss, Inc.     

Investigations of the rhodopsin/Meta I and rhodopsin/Meta II transitions of bovine rod outer segments by means of kinetic infrared spectroscopy

We have applied our recently developed technique of flash induced kinetic infrared spectroscopy to the rhodopsin/Meta I and rhodopsin/Meta II transitions. Features of the infrared spectrum reflecting the C=C-vibration and the isomeric form of the chromophore are in agreement with resonant Raman experiments. Different results are obtained for the C=N-vibration of the Schiff base retinal opsin link. They are interpreted in terms of a Schiff base protonated via an hydrogen bond. A proton transfer in the excited state is suggested to explain the deviating results. In addition we have obtained spectral changes which cannot be attributed to molecular changes in the chromophore. We assume that these spectral features reflect molecular events in the protein part of rhodopsin.     

Evidence for a driving role of ingrowing axons for the shifting of older retinal terminals in the tectum of fish

In amphibians and teleosts, retina and tectum grow incongruently. In order to maintain the retinotopy of the retinotectal projection, Gaze, Keating, and Chung (1974) postulated a shifting of terminals throughout growth. In order to test the possibility that ingrowing retinal fibers are the driving force for this shifting, we induced a permanent retinal projection into the ipsilateral tectum in juveniles of the cichlid fish Haplochromis burtoni. The surface of the tectum had increased (11–18 months later) 2.5–5.8 times, and the surface of the retina 8.6–14 times. Filling of ganglion cells with horseradish peroxidase (HRP) retrogradely from the tectum showed ipsilaterally regenerating ganglion cells only in the center of the retina. The position of ganglion cells indicated that the ipsilateral projection derived only from axotomized and regenerating retinal ganglion cells but not from those newly born. Ipsilaterally projecting retinal fibers showed terminals only in the rostral half of the tectum. Comparison of area of terminations of ipsilaterally projecting ganglion cells at various times after the crush provided no evidence for expansion or a shift into caudal tectal areas throughout the period of growth. These findings are compatible with the idea that newly ingrowing fibers induce older terminals to move caudally.     

Light-driven primary sodium ion transport in halobacterium halobium membranes

Light-induced sodium extrusion from H halobium cell envelope vesicles proceeds largely through an uncoupler-sensitive pathway involving bacteriorhodopsin and a proton/sodium antiporter. Vesicles from bacteriorhodopsin-negative strains also extrude sodium ions during illumination, but this transport is not sensitive to uncouplers and has been proposed to involve a light-energized primary sodium pump. Proton uptake in such vesicles is passive, and under steady-state illumination the large electrical potential (negative inside) is just balanced by a pH difference (acid inside), so that the protonmotive force is near zero. Action spectra indicated that this effect of illumination is attributable to a pigment absorbing near 585 nm (of 568 for bacteriorhodopsin). Bleaching of the vesicles by prolonged illumination with hydroxylamine results in inactivation of the transport; retinal addition causes partial return of the activity. Retinal addition also causes the appearance of an absorption peak at 588 nm, while the absorption of free retinal decreases. The 588 nm pigment is present in very small quantities (0.13 nmole/mg protein), and behaves differently from bacteriorhodopsin in a number of respects. Vesicles can be prepared from bacteriorhodopsin-containing H halobium strains in which primary transport for both protons and sodium can be observed. Both pumps appear to cause the outward transport of the cations. The observations indicate the existence of a second retinal protein, in addition to bacteriorhodopsin, in H halobium, which is associated with primary sodium translocation. The initial proton uptake normally observed during illumination of whole H halobium cells may therefore be a passive flux in response to the primary sodium extrusion.